SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Primary nasal influenza infection rewires tissue-scale memory response dynamics.

The nasal mucosa is often the initial site of respiratory viral infection, replication, and transmission. Understanding how infection shapes tissue-scale primary and memory responses is critical for designing mucosal therapeutics and vaccines. We generated a single-cell RNA-sequencing atlas of the murine nasal mucosa, sampling three regions during primary influenza infection and rechallenge. Compositional analysis revealed restricted infection to the respiratory mucosa with stepwise changes in immune and epithelial cell subsets and states. We identified and characterized a rare subset of Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells, which concurrently increased with tissue-resident memory T (TRM)-like cells. Proportionality analysis, cell-cell communication inference, and microscopy underscored the CXCL16-CXCR6 axis between KNIIFE and TRM cells. Secondary influenza challenge induced accelerated and coordinated myeloid and lymphoid responses without epithelial proliferation. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses.

Are we there yet? An immune field trip through human embryonic development.

Technical, analytical, and ethical challenges have obscured our understanding of immune cell subset ontogeny during human fetal development. Recently published in Science, Suo et al. (2022) apply multiple single-cell and spatial tools to provide a comprehensive roadmap during human gestation.

Evolution and Diversity of Immune Responses during Acute HIV Infection.

Understanding the earliest immune responses following HIV infection is critical to inform future vaccines and therapeutics. Here, we review recent prospective human studies in at-risk populations that have provided insight into immune responses during acute infection, including additional relevant data from non-human primate (NHP) studies. We discuss the timing, nature, and function of the diverse immune responses induced, the onset of immune dysfunction, and the effects of early anti-retroviral therapy administration. Treatment at onset of viremia mitigates peripheral T and B cell dysfunction, limits seroconversion, and enhances cellular antiviral immunity despite persistence of infection in lymphoid tissues. We highlight pertinent areas for future investigation, and how application of high-throughput technologies, alongside targeted NHP studies, may elucidate immune response features to target in novel preventions and cures.

Germline-Encoded Affinity for Cognate Antigen Enables Vaccine Amplification of a Human Broadly Neutralizing Response against Influenza Virus.

Antibody paratopes are formed by hypervariable complementarity-determining regions (CDRH3s) and variable gene-encoded CDRs. The latter show biased usage in human broadly neutralizing antibodies (bnAbs) against both HIV and influenza virus, suggesting the existence of gene-endowed targeting solutions that may be amenable to pathway amplification. To test this, we generated transgenic mice with human CDRH3 diversity but simultaneously constrained to individual user-defined human immunoglobulin variable heavy-chain (VH) genes, including IGHV1-69, which shows biased usage in human bnAbs targeting the hemagglutinin stalk of group 1 influenza A viruses. Sequential immunization with a stalk-only hemagglutinin nanoparticle elicited group 1 bnAbs, but only in IGHV1-69 mice. This VH-endowed response required minimal affinity maturation, was elicited alongside pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. These results indicate that the human repertoire could, in principle, support germline-encoded bnAb elicitation using a single recombinant hemagglutinin immunogen.

Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion.

CD8+ T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8+ T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor ß (TCRß) analysis revealed that class II-restricted CD8+ T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8+ T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8+ T cell responses can exist in a chronic human viral infection, and may contribute to immune control.

Innate Lymphoid Cells Are Depleted Irreversibly during Acute HIV-1 Infection in the Absence of Viral Suppression.

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction.

Publisher Correction: Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

Tuberculosis is the leading cause of death by an infectious disease worldwide1. However, the involvement of innate lymphoid cells (ILCs) in immune responses to infection with Mycobacterium tuberculosis (Mtb) is unknown. Here we show that circulating subsets of ILCs are depleted from the blood of participants with pulmonary tuberculosis and restored upon treatment. Tuberculosis increased accumulation of ILC subsets in the human lung, coinciding with a robust transcriptional response to infection, including a role in orchestrating the recruitment of immune subsets. Using mouse models, we show that group 3 ILCs (ILC3s) accumulated rapidly in Mtb-infected lungs and coincided with the accumulation of alveolar macrophages. Notably, mice that lacked ILC3s exhibited a reduction in the accumulation of early alveolar macrophages and decreased Mtb control. We show that the C-X-C motif chemokine receptor 5 (CXCR5)-C-X-C motif chemokine ligand 13 (CXCL13) axis is involved in Mtb control, as infection upregulates CXCR5 on circulating ILC3s and increases plasma levels of its ligand, CXCL13, in humans. Moreover, interleukin-23-dependent expansion of ILC3s in mice and production of interleukin-17 and interleukin-22 were found to be critical inducers of lung CXCL13, early innate immunity and the formation of protective lymphoid follicles within granulomas. Thus, we demonstrate an early protective role for ILC3s in immunity to Mtb infection.

Allergic inflammatory memory in human respiratory epithelial progenitor cells.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases1. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway2-4. Allergic inflammation can develop from persistent activation5 of type 2 immunity6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps7. Basal cell hyperplasia is a hallmark of severe disease7-9, but it is not known how these progenitor cells2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing12, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic13, epigenetic14,15 and extrinsic factors11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas-based knockouts and RNA-interference-based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.

Probing the relative orientation of molecules bound to DNA through controlled interference using second-harmonic generation.

A method is described in which the interference of radiated second-harmonic electric fields generated by a pair of oriented molecules intercalated into double-stranded DNA is controlled and measured. The results show that the relative molecular orientation of the two molecules significantly changes the magnitude of the observed second-harmonic generation intensity, which is described by a simple model that accounts for the interferences of the radiated fields. The technique presented shows promise for future experiments investigating structural changes induced by the formation of a DNA-biomolecule complex.

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation.

The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg(2+), the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg(2+), which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way.

Pro-inflammatory feedback loops define immune responses to pathogenic Lentivirus infection.

BACKGROUND: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. METHODS: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. RESULTS: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. CONCLUSIONS: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Primary nasal viral infection rewires the tissue-scale memory response.

The nasal mucosa is frequently the initial site of respiratory viral infection, replication, and transmission. Recent work has started to clarify the independent responses of epithelial, myeloid, and lymphoid cells to viral infection in the nasal mucosa, but their spatiotemporal coordination and relative contributions remain unclear. Furthermore, understanding whether and how primary infection shapes tissue-scale memory responses to secondary challenge is critical for the rational design of nasal-targeting therapeutics and vaccines. Here, we generated a single-cell RNA-sequencing (scRNA-seq) atlas of the murine nasal mucosa sampling three distinct regions before and during primary and secondary influenza infection. Primary infection was largely restricted to respiratory mucosa and induced stepwise changes in cell type, subset, and state composition over time. Type I Interferon (IFN)-responsive neutrophils appeared 2 days post infection (dpi) and preceded transient IFN-responsive/cycling epithelial cell responses 5 dpi, which coincided with broader antiviral monocyte and NK cell accumulation. By 8 dpi, monocyte-derived macrophages (MDMs) expressing Cxcl9 and Cxcl16 arose alongside effector cytotoxic CD8 and Ifng-expressing CD4 T cells. Following viral clearance (14 dpi), rare, previously undescribed Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells expressing multiple genes with immune communication potential increased concurrently with tissue-resident memory T (TRM)-like cells and early IgG+/IgA+ plasmablasts. Proportionality analysis coupled with cell-cell communication inference, alongside validation by in situ microscopy, underscored the CXCL16-CXCR6 signaling axis between MDMs and effector CD8 T cells 8dpi and KNIIFE cells and TRM cells 14 dpi. Secondary influenza challenge with a homologous or heterologous strain administered 60 dpi induced an accelerated and coordinated myeloid and lymphoid response without epithelial proliferation, illustrating how tissue-scale memory to natural infection engages both myeloid and lymphoid cells to reduce epithelial regenerative burden. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses upon rechallenge.

An enhanced IL17 and muted type I interferon nasal epithelial cell state characterizes severe COVID-19 with fungal coinfection.

Recent case reports and epidemiological data suggest that fungal infections represent an underappreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing data set characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our previous study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL17 stimulation and anti-fungal immunity. Further, we observe a significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggest that IL17 stimulation-in part driven by Candida colonization-and blunted interferon signaling represent a common feature of severe COVID-19 infection. IMPORTANCE: In this paper, we present an analysis suggesting that symptomatic and asymptomatic fungal coinfections can impact patient disease progression during COVID-19 hospitalization. By looking into the presence of other pathogens and their effect on the host immune response during COVID-19 hospitalizations, we aim to offer insight into an underestimated scenario, furthering our current knowledge of determinants of severity that could be considered for future diagnostic and intervention strategies.

Well-defined nanofibers with tunable morphology from spherical colloidal building blocks.

From particles to fibers: Nanofibers with different morphologies and periodicities can be fabricated by supraparticular assembly of magnetic spherical nanoparticles. A linear sintering process is used to merge the assembled colloids together. The structure of the obtained fibers is controlled by the process parameters and the morphology of the spherical colloidal building blocks.

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer.

Introduction: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). Methods: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). Results: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. Conclusions: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors.

HIV specific CD8+ TRM-like cells in tonsils express exhaustive signatures in the absence of natural HIV control.

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue-resident memory (TRM) CD8+ T-cells is of great interest, but limited data exist on TRM-like cells within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and a transcriptionally TRM-like profile distinct from blood CD8+ T-cells. In PLWH, CD8+ TRM-like cells are expanded and adopt a more cytolytic, activated, and exhausted phenotype not reversed by antiretroviral therapy (ART). This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood suggesting a higher antigen burden in tonsils. Single-cell transcriptional and clonotype resolution showed that these HIV-specific CD8+ T-cells in the tonsils express heterogeneous signatures of T-cell activation, clonal expansion, and exhaustion ex-vivo. Interestingly, this signature was absent in a natural HIV controller, who expressed lower PD-1 and CXCR5 levels and reduced transcriptional evidence of T-cell activation, exhaustion, and cytolytic activity. These data provide important insights into lymphoid tissue-derived HIV-specific CD8+ TRM-like phenotypes in settings of HIV remission and highlight their potential for immunotherapy and targeting of the HIV reservoirs.

MR1-Restricted MAIT Cells From The Human Lung Mucosal Surface Have Distinct Phenotypic, Functional, and Transcriptomic Features That Are Preserved in HIV Infection.

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αß T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.